Chloride (CI) currents contribute to agonist-induced depolarization of vascular smooth muscle (VSM) cells. As part of the original grant proposal for which this application is a continuation, we created a mouse lacking a specific chloride channel, CIC-3 (encoded by the CIcn3 gene). Five findings from our laboratory demonstrate the importance of CIC-3 to cardiovascular function: 1) Animals lacking CIC-3 CI channels display multiple cardiovascular abnormalities including; hypertension, left ventricular hypertrophy and diastolic dysfunction, and impaired endothelium-dependent relaxation in resistance vessels, 2) CIC-3 channels are located intracellularly in resting VSM cells, but are inserted into the plasma membrane in response to All, 3) there is an unappreciated diversity of CIC-3 protein structure resulting from alternative splicing that may alter membrane trafficking, 4) Clcn3-/- cells have increased levels of intracellular reactive oxygen species (ROS), and 5) superoxide anion may pass through CIC-3 channels. There is conflicting data in the literature as to the biophysical nature of CIC-3. These inconsistencies may reflect a lack of in-depth knowledge of channel localization within the cell and the mechanisms that move the channel between cellular compartments. We will first carefully define the localization and trafficking of CIC-3. We will then test the hypothesis that the altered microvascular function observed in Clcn3-/- mice is related to the absence of a conductance that normally provides a mechanism by which superoxide anion moves across biological membranes. In this renewal application, we will; 1) Define the subcellular localization of native CIC-3 protein in murine VSM and identify factors that regulate the trafficking of CIC-3 to the plasma membrane in response to angiotensin II, 2) Define the subcellular localization of the six distinct splice variants of CIC-3 in VSM cells using FIV-driven expression of recombinant CIC-3 protein and identify motifs and physiologic factors that regulate membrane trafficking of CIC-3, and 3) Determine why intracellular ROS levels are elevated in Clcn3-/- cells and tissues and discern if this increase is physiologically relevant. The CIC-3 knockout mouse represents a novel single-gene defect model of hypertension. Careful analysis of the physiological defects in Clcn3-/- mice will yield important insight into cellular ROS metabolism and the link between ROS and high blood pressure.